Development of nanosupplements based on bioactive compound encapsulation and their incorporation into diet constitutes a field that is growing exponentially. Among flavonoids, chrysin (Chrys) shows high antitumor activity but its poor water solubility limits its applications. Bovine serum albumin nanoparticles (BSAnp) with sizes in the range of 13-28 nm were previously prepared by applying a heat-induced self-assembly method, and they were able to load Chrys. Hence, the aim of this study is to provide information about elaboration of Chrys nanosupplements considering the incidence of the freeze-drying process of Chrys-loaded and unloaded BSAnp systems on aqueous redispersibility and on the retention of their physicochemical properties as examined by DSC, turbidity and DLS measurements. In general, the physicochemical properties of Chrys-loaded BSAnp were retained after the freeze-drying process. The cytotoxic activity of the reconstituted powders was assayed on MCF-7 and MDA-MB-231 cell lines (as models of cancer cell lines) by using the MTT assay. Furthermore, the mechanism of cell death was researched by Annexin V-FITC flow cytometry. The apoptosis-mediated cytotoxicity was higher for Chrys-loaded BSAnp than for the BSAnp system. The greatest in vitro cytotoxic effect was observed for the smallest sized Chrys-loaded BSAnp, which was obtained at pH 11.0 and 70 °C. Finally, this system was subjected to static in vitro gastrointestinal digestion using the standardized protocol, and ∼14% Chrys was released from BSAnp after digestion. These results would provide a procedure to obtain bioactive nanosupplements in a soluble form, and they would suggest that smaller sized Chrys-loaded BSAnp could be a promising oral delivery system for potential cancer therapies.